Safeguarding American Agriculture 

Federal Relay Service 

APHIS is an agency of USDA’s Marketing and Regulatory Programs (Voice/TTY/ASCII/Spanish)

An Equal Opportunity Provider and Employer 1-800-877-8339 

March 29, 2012 

CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 12-06 

TO: Biologics Licensees, Permittees, and Applicants 

 Directors, Center for Veterinary Biologics 

 Veterinary Services Leadership Team 

 

FROM: Richard E. Hill, Jr. /s/ Richard E. Hill, Jr.

 Director 

 Center for Veterinary Biologics 

SUBJECT: Detection of Mycoplasma Contamination in Vaccines and Biological 

Products

I. PURPOSE 

The Center for Veterinary Biologics (CVB) is validating a Polymerase Chain Reaction 

(PCR) detection assay as an alternative for the agar culture method specified in Title 9, 

Code of Federal Regulations (9 CFR), Part 113.28, Detection of Mycoplasma 

Contamination, and is planning a field study as part of the validation. 

II. BACKGROUND 

Veterinary biologics, viral master seeds, master cell stocks, and ingredients of animal 

origin used in the production of veterinary biologics must be shown to be free of 

contaminating mycoplasma, following the procedure specified in 9 CFR 113.28. This 

test involves a broth culture step for the amplification of low levels of viable 

mycoplasma, followed by an agar plating step for the detection of mycoplasma colonies. 

The test procedure differs from assays required by regulatory agencies of other countries; 

consequently, products exported to certain countries must be tested by both 9 CFR113.28 

and by the method accepted by the importing country. 

With the aim of harmonization of the general requirements for mycoplasma 

contamination testing, the International Cooperation on Harmonization of Technical 

Requirements for Registration of Veterinary Medicinal Products (VICH) has developed 

draft guideline GL34, “Testing for the detection of mycoplasma contamination”. This 

document provides for the option of using a validated nucleic acid amplification 

methodology, provided that the detection limit of the assay is at least equivalent to that of 

the culture methods listed in GL34. Upon approval of the draft guideline, the guideline 

will be recommended for adoption by the CVB. 

United States 

Department of 

Agriculture 

Animal and Plant 

Health Inspection 

Service 

Veterinary Services 

Center for Veterinary 

Biologics 

1920 Dayton Avenue 

PO Box 844 

Ames, IA 50010 

(515) 337- 6100 
CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 12-06 

Page 2 

III. ACTION 

The CVB has developed a PCR assay for the detection of mycoplasma, and is currently in 

the process of validating the method for implementation at the CVB. The PCR assay 

does not replace the 9 CFR 113.28; it is intended for use with broth enrichment, replacing 

the agar detection step of 9 CFR 113.28. The CVB laboratory is currently using the PCR 

assay to screen viral Master Seeds and Master Cell Stocks, and is running the PCR assay 

in parallel with the 9 CFR 113.28 procedure for routine mycoplasma testing. 

The CVB is soliciting help from firms willing to participate in a field study to evaluate 

the reproducibility of the PCR method. Participating firms will be asked to test the PCR 

detection method side-by-side with the 9 CFR 113.28 agar detection method, using the 

five mycoplasma strains that are listed in GL34. The CVB will provide a detailed study 

protocol and will supply the mycoplasma strains, PCR protocol, and positive control. 

Firms wishing to incorporate the PCR assay into their mycoplasma testing may use the 

data they generate in the field study as part of their validation of the assay. 

IV. IMPLEMENTATION/ APPLICABILITY 

Requests for additional information should be directed to Dr. Geetha Srinivas at 

Geetha.B.Srinivas@aphis.usda.gov.